Metalloproteome Factory: From Crude Extract to Identification of Metalloproteins via 3-Dimensional HPLC-ICP-MS

Metalloproteins are essential for a vast array of biological processes in all living organisms. Identification of multiple metalloproteins from native expression ("metalloproteomics") based on a single chromatography has low resolution and/or sensitivity, while classical protein purification lacks the versatility required for purifying multiple proteins for proteomics. We present a new metalloproteomics method based on three dimensions of metal-free liquid chromatography (HPLC) hyphenated online with inductively coupled sector-field mass spectrometry (ICP-sfMS): anion exchange chromatography (AEC) followed by either chromatofocusing (CF) or hydrophobic interaction chromatography (HIC) and finally size exclusion chromatography (SEC). We used this 3D technique to purify naturally abundant metalloproteins from crude extracts mainly from plants but also from bacteria to a quantity and purity sufficient for protein identification and quantification by mass spectrometry (Tims-TOF-MS). Protein mass spectrometry data are available via ProteomeXchange with identifier PXD056024. Sensitivity and signal/background ratio were sufficient to identify proteins at concentrations <1 nM and total amounts <1 pmol in the final SEC. As shown by application examples, the method can be immediately applied to various biological materials from a large range of organisms without individual optimization for single proteins or organisms. It can thus be used to resolve mechanisms of metal metabolism in various contexts, e.g., metal deficiency, nutrition, toxicity, and detoxification.